title: The adipophilin C terminus is a self-folding membrane-binding domain that is important for milk lipid secretion.
abstract: Cytoplasmic lipid droplets (CLD) in mammary epithelial cells undergo secretion by a unique membrane envelopment process to produce milk lipids. Adipophilin (ADPH/Plin2), a member of the perilipin/PAT family of lipid droplet-associated proteins, is hypothesized to mediate CLD secretion through interactions with apical plasma membrane elements. We found that the secretion of CLD coated by truncated ADPH lacking the C-terminal region encoding a putative four-helix bundle structure was impaired relative to that of CLD coated by full-length ADPH. We used homology modeling and analyses of the solution and membrane binding properties of purified recombinant ADPH C terminus to understand how this region possibly mediates CLD secretion. Homology modeling supports the concept that the ADPH C terminus forms a four-helix bundle motif and suggests that this structure can form stable membrane bilayer interactions. Circular dichroism and protease mapping studies confirmed that the ADPH C terminus is an independently folding α-helical structure that is relatively resistant to urea denaturation. Liposome binding studies showed that the purified C terminus binds to phospholipid membranes through electrostatic dependent interactions, and cell culture studies documented that it localizes to the plasma membrane. Collectively, these data provide direct evidence that the ADPH C terminus forms a stable membrane binding helical structure that is important for CLD secretion. We speculate that interactions between the four-helix bundle of ADPH and membrane phospholipids may be an initial step in milk lipid secretion.
authors.affiliation: Graduate Program in Molecular Biology, Division of Basic Reproductive Science, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045, USA.
authors.forename: Brandi M
authors.initials: BM
authors.lastname: Chong
authors.forename: Tanya D
authors.initials: TD
authors.lastname: Russell
authors.forename: Jerome
authors.initials: J
authors.lastname: Schaack
authors.forename: David J
authors.initials: DJ
authors.lastname: Orlicky
authors.forename: Philip
authors.initials: P
authors.lastname: Reigan
authors.forename: Mark
authors.initials: M
authors.lastname: Ladinsky
authors.forename: James L
authors.initials: JL
authors.lastname: McManaman
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D005260:Female; D007774:Lactation; D008321:Mammary Glands, Animal / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008567:Membranes, Artificial; D051379:Mice; D008892:Milk / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D000070780:Perilipin-2; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D008565:Membrane Proteins; D008567:Membranes, Artificial; D000070780:Perilipin-2; D010743:Phospholipids; C000602983:Plin2 protein, mouse